Reactor for the quantitative analysis of necleic acids

ABSTRACT

A reactor for the quantitative analysis of target nucleic acids using an evanescent wave detection technique and a method of use thereof is provided. The reactor includes a substrate with a cavity, a buffer layer arranged over the substrate; a cover plate arranged over the buffer layer, and inlet and outlet ports. The reactor is thermally and chemically stable for PCR processing and suitable for an evanescent wave detection technique.

BACKGROUND OF THE INVENTION

An important technique currently used in bioanalysis and in the emergingfield of genomics is the polymerase chain reaction (PCR) amplificationof DNA. As a result of this powerful tool, it is possible to start withotherwise undetectable amounts of DNA and create ample amounts of thematerial for subsequent analysis. PCR uses a repetitive series of stepsto create copies of polynucleotide sequences located between twoinitiating (“primer”) sequences. Starting with a template, two primersequences (usually about 15-30 nucleotides in length), PCR buffer, freedeoxynucleoside tri-phosphates (dNTPs), and thermostable DNA polymerase(commonly TAQ polymerase from Thermus aquaticus), these components aremixed, and heated to separate the double-stranded DNA. A subsequentcooling step allows the primers to anneal to complementary sequences onsingle-stranded DNA molecules containing the sequence to be amplified.Replication of the target sequence is accomplished by the DNApolymerase, which produces a strand of DNA that is complementary to thetemplate. Repetition of this process doubles the number of copies of thesequence of interest, and multiple cycles increase the number of copiesexponentially.

Since PCR requires repeated cycling between higher and lowertemperatures, PCR devices must be fabricated from materials capable ofwithstanding such temperature changes. The materials must bemechanically and chemically stable at high temperatures, and capable ofwithstanding repeated temperature changes without mechanicaldegradation. Furthermore, the materials must be compatible with the PCRreaction itself, and not inhibit the polymerase or bind DNA.

Conventional PCR is typically carried out in tubes, microplates, andcapillaries, all of which could be sealed conveniently. However, thegeometry of these tubes, microplates, and capillaries render them notsuitable for evanescent wave detection methods.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention may be best understood by referring to thefollowing description and accompanying drawings, which illustrate suchembodiments. In the drawings:

FIG. 1 illustrates a view of a cartridge capable of evanescent wavedetection of fluorescently tagged amplicons in a microarrayed PCRprocess.

FIG. 2 illustrates a side view of a cartridge capable of evanescent wavedetection of fluorescently tagged amplicons in a microarrayed PCRprocess.

FIG. 3 illustrates a view of another cartridge capable of evanescentwave detection of fluorescently tagged amplicons in a microarrayed PCRprocess.

FIG. 4 illustrates a side view of another cartridge capable ofevanescent wave detection of fluorescently tagged amplicons in amicroarrayed PCR process.

DEFINITIONS

As used herein, certain terms have the following meanings. All otherterms and phrases used in this specification have their ordinarymeanings as one of skill in the art would understand. Such ordinarymeanings may be obtained by reference to technical dictionaries, such asHawley's Condensed Chemical Dictionary 11^(th) Edition, by Sax andLewis, Van Nostrand Reinhold, New York, N.Y., 1987, and The Merck Index,11^(th) Edition, Merck & Co., Rahway N.J. 1989.

As used herein, the term “and/or” means any one of the items, anycombination of the items, or all of the items with which this term isassociated.

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Therefore, forexample, a reference to “a formulation” includes a plurality of suchformulations, so that a formulation of compound X includes formulationsof compound X.

As used herein, the term “about” means a variation of 10 percent of thevalue specified, for example, about 50 percent carries a variation from45 to 55 percent. For integer ranges, the term about can include one ortwo integers greater than and less than a recited integer.

As used herein, the term “amplicons” refers to the products ofpolymerase chain reactions (PCR). Amplicons are pieces of DNA that havebeen synthesized using amplification techniques (e.g., a double-strandedDNA with two primers). The amplicon may contain, for example, a primertagged with a fluorescent molecule at the 5′ end.

As used herein, the terms “array” and “microarray” refer to anarrangement of elements (i.e., entities) into a material or device. Inanother sense, the term “array” refers to the orderly arrangement (e.g.,rows and columns) of two or more assay regions on a substrate.

As used herein, the term “evanescent” refers to a nearfield standingwave exhibiting exponential decay with distance. As used in optics,evanescent waves are formed when sinusoidal waves are internallyreflected off an interface at an angle greater than the critical angleso that total internal reflection occurs.

As used herein, the term “hybridization” refers to the pairing ofcomplementary nucleic acids.

As used herein, the term “motive force” is used to refer to any meansfor inducing movement of a sample along a flow path in a reactor, andincludes application of an electric potential across any portion of thereactor, application of a pressure differential across any portion ofthe reactor or any combination thereof.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule including, but not limited to, DNA or RNA.

As used herein, the term “optical detection path” refers to aconfiguration or arrangement of detection means to form a path wherebyelectromagnetic radiation is able to travel from an external source to ameans for receiving radiation, wherein the radiation traverses thereaction chamber.

As used herein, the term “polymerase chain reaction” (PCR) refers to themethod of K. B. Mullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and4,965,188.

As used herein, the term “reactor” refers to a device, which can be usedin any number of chemical processes involving a fluid. The primaryprocess of interest is the amplification of DNA using the polymerasechain reaction. Optionally, DNA amplification may be conducted alongwith one or more other types of procedures.

As used herein, the term “stability” refers to the ability of a materialto withstand deterioration or displacement and to provide reliabilityand dependability.

As used herein, the term “substrate” refers to material capable ofsupporting associated assay components (e.g., assay regions, cells, testcompounds, etc.).

As used herein, the term “target nucleic acid” refers to apolynucleotide inherent to a pathogen that is to be detected. Thepolynucleotide is genetic material including, for example, DNA/RNA,mitochondrial DNA, rRNA, tRNA, mRNA, viral RNA, and plasmid DNA.

As used herein, the term “water impermeable” refers to a material inwhich water will not pass through the material.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a reactor for the quantitative analysisof target nucleic acids using an evanescent wave detection technique anda method of use thereof. The reactor includes a substrate with a cavity,a buffer layer arranged over the substrate; a cover plate arranged overthe buffer layer, and inlet and outlet ports. The reactor is thermallyand chemically stable for PCR processing and suitable for an evanescentwave detection technique.

The PCR process that occur inside the reactor require specialtemperature conditions, such as a circular cycle of high and lowtemperatures. The temperature change of the liquid and the reactionchamber are regulated by a heating and cooling system.

At high temperatures, the sample liquid expands and increases thepressure inside the reaction chamber. Conversely, at low temperatures,the sample liquid shrinks and decreases the pressure inside the reactionchamber. Any deformation of the reaction chamber will cause incompleteadherence between the cover layer and the substrate and result inleakage. In the case of PCR amplification, even a small amount of littleleakage may result in false positives. To prevent this leakage, a bufferlayer is used.

For real-time quantitative analysis of target nucleic acids, severalmethods utilizing evanescent wave detection techniques have beendisclosed including, for example, the techniques described in Xu (U.S.Patent Application Publication No. 2006/0088844) and in PCT PatentApplication Serial No. PCT/CN2007/003124, entitled “A QUANTITATIVEMETHOD FOR OLIGONUCLEOTIDE MICROARRAY” filed Nov. 5, 2007.

In these methods describing the real-time quantitative analysis oftarget nucleic acids, the target nucleic acids in the sample areamplified using the polymerase chain reaction (PCR). PCR is begun byplacing the target nucleic acids in a buffer containing the nucleotidesadenine (A), thymine (T), cytosine (C) and guanine (G) (collectivelyreferred to as dNTPs), a DNA polymerase, and primers. The primers areshort strands of DNA, with sequences that complement specific regions ofthe target nucleic acids. The primers initiate replication of the targetnucleic acids. The primers may be fluorescently tagged with fluorescentmolecules at the 5′ end or the dNTPs are fluorescently tagged.

This type of PCR process has three main steps: denaturation, annealingand extension. In the denaturation step, the mixture is heated to about94° C. (Centrigrade), at which point the target DNA separates intosingle strands. The mixture is quickly cooled. As the temperature fallsto about 60° C., the annealing step occurs, in which the primers, whichare fluorescently tagged, hybridize or bind to their complementarysequences on the target nucleic acids. The extension step may beperformed at about 60° C. or may be raised to the 72-78° C. range. Inthis step, the DNA polymerase uses the dNTPs in solution to extend theannealed primers, which are fluorescently tagged, and forms new strandsof DNA known as an amplicons. The mixture is briefly reheated back toabout 94° C. to separate the newly created double helix stands intosingle strands of nucleic acid, which begins another cycle of the PCRprocess. With each cycle of the PCR process, the number of copies of theoriginal target nucleic acids roughly doubles.

The PCR buffer may additionally contain fluorescently tagged primers,that is, primers having a fluorescent dye molecule attached to them, sothat upon completion of each PCR cycle, the amplicons produced arefluorescently tagged. The amplicons of the target nucleic acids arelocalized, using probe strands of DNA known as target nucleic acidprobes. The target nucleic acid probes have the same complementary,nucleotide sequence as the target nucleic acids. The target nucleic acidprobes are tethered to a substrate surface in a known, two-dimensionalpattern, with the substrate surface forming part of the reaction cellcontaining the PCR ingredients.

The PCR buffer may also include coating agents or surfactants to preventnon-specific binding by modifying the interior surfaces of the reactor.Examples of such coating agents include polyethylene oxide triblockcopolymers, polyethylene glycols (PEG) having molecular weights rangingfrom about 200 to about 8000, natural polymers such as bovine serumalbumen (BSA) or any other moieties that provide the desired surfacecharacteristics, particularly those that reduce the sorption ofbiomolecules such as proteins and nucleic acid

A solution containing the sample to be amplified and appropriate buffersand reagents is typically introduced into the reactor via anyappropriate methodology. Introduction of the sample may be achievedusing any convenient means, including electrokinetic injection,hydrodynamic injection, spontaneous fluid displacement and the like. Theparticular means employed will, for the most part, depend on theconfiguration of the channel as well as the necessity to introduce aprecise volume of sample.

During the annealing and extension phases of the PCR process, the targetamplicons hybridize to their corresponding target nucleic acid probes.The hybridized, fluorescently tagged amplicons are illuminated with anevanescent wave of light of the appropriate wavelength to activate thefluorescent dye molecules of the fluorescently tagged primers or thefluorescently tagged dNTPs. This evanescent wave decays exponentially inpower after entering the reaction cell via the substrate surface towhich the target nucleic acid probes are tethered, with an effectivepenetration range of about 300 mu. This means that the evanescent wavepenetrates far enough into the reaction cell to activate thefluorescently tagged amplicons hybridized to those target nucleic acidprobes, but that it does not activate the fluorescently tagged molecules(e.g., the fluorescently tagged primers or the fluorescently taggeddNTPs) in solution in the main body of the reaction cell. By monitoringthe strength of the fluorescence at various locations on the substratesurface, the current abundance of amplicons of the corresponding targetnucleic acids can be determined. The results are used to obtain aquantitative measure of the abundance of a specific target in theoriginal sample, in a manner analogous to the real-time PCR calculation.

The Reactor

In an embodiment, FIG. 1 schematically illustrates a reactor that can beused in conducting a chemical process such as PCR. The device isgenerally represented at 11, comprising substrate 13 having a planarsurface 15 and containing a cavity 17. A buffer layer 19 is shownarranged over the planar surface 15 of substrate 13. A cover plate 21 isshown arranged over the top surface 23 of the buffer layer 19.

Prior to use of the device, the underside 25 of the cover plate 21 isaligned with and placed against the top surface 23 of the buffer layer19 on the planar surface 15 of substrate 13 (see, e.g., FIG. 2). Thecover plate 21, in combination with the buffer layer 19, and cavity 17,form a reaction chamber in which the desired chemical process is carriedout. Fluid, e.g., sample to be analyzed, analytical reagents, reactantsor the like, are introduced into the reaction chamber from an externalsource through inlet port 27. The outlet port 29 enables passage offluid from the reaction chamber to an external receptacle. Accordingly,the reactor is closed by aligning the cover plate 21 with the bufferlayer 19 on substrate 13, forming a seal. In some embodiments, thebuffer layer 19 is not cured. In other embodiments, the buffer layer 19is cured. This seal results in formation of a reaction chamber intowhich fluids may be introduced through inlet port 27 and removed throughoutlet port 29. A set of plugs (e.g., rubber) with the proper size,hardness, and chemical resistance may be used to seal the inlet port 27and outlet port 29 of the reaction chamber.

In another embodiment, FIG. 3 schematically illustrates a reactor thatcan be used in conducting a chemical process such as PCR. The device isgenerally represented at 11, comprising substrate 13 having a planarsurface 15 and containing a cavity 17. A buffer layer 19 is shownarranged over the planar surface 15 of substrate 13. A cover plate 21 isshown arranged over the top surface 23 of the buffer layer 19.

Prior to use of the device, the underside 25 of the cover plate 21 isaligned with and placed against the top surface 23 of the buffer layer19 on the planar surface 15 of substrate 13 (see, e.g., FIG. 4). Thecover plate 21, in combination with the buffer layer 19, and cavity 17,form a reaction chamber in which the desired chemical process is carriedout. Fluid, e.g., sample to be analyzed, analytical reagents, reactantsor the like, are introduced into the reaction chamber from an externalsource through inlet port 27. The outlet port 29 enables passage offluid from the reaction chamber to an external receptacle. Accordingly,the reactor is closed by aligning the cover plate 21 with the bufferlayer 19 on substrate 13, forming a seal. In some embodiments, thebuffer layer 19 is not cured. In other embodiments, the buffer layer 19is cured. This seal results in formation of a reaction chamber intowhich fluids may be introduced through inlet port 27 and removed throughoutlet port 29. A set of plugs (e.g., rubber) with the proper size,hardness, and chemical resistance may be used to seal the inlet port 27and outlet port 29 of the reaction chamber.

The Substrate and Cover Plate

The materials used to form the substrates and cover plates in theembodiments are selected with regard to physical and chemicalcharacteristics that are desirable for a particular application. Thesubstrate and cover plates should be chemically inert and physicallystable with respect to any reagents with which they comes into contact,under the reaction conditions used (e.g., with respect to pH, electricfields, etc.). Since PCR involves relatively high temperatures, it isimportant that all materials be chemically and physically stable withinthe range of temperatures used. For use with optical detection means,the materials used should be optically transparent, typicallytransparent to wavelengths in the range of about 150 nm to 800 nm.

For example, in some embodiments, the substrate includes a planar (i.e.,2 dimensional) glass, metal, composite, plastic, silica, or otherbiocompatible or biologically unreactive composition. Many substratesmay be employed. The substrate may be biological, nonbiological,organic, inorganic, or a combination of any of these, existing asparticles, strands, precipitates, gels, sheets, tubing, spheres,containers, capillaries, pads, slices, films, plates, slides, etc. Thesubstrate may have any convenient shape, such as a disc, square, sphere,circle, etc. The substrate is generally flat but may take on a varietyof alternative surface configurations. For example, the substrate maycontain raised or depressed regions on which the synthesis takes place.The substrate and its surface can form a rigid support on which to carryout the reactions described herein. The substrate and its surface arealso chosen to provide appropriate light-absorbing characteristics. Forinstance, the substrate may be a polymerized Langmuir Blodgett film, aglass, a functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modifiedsilicon, or any one of a wide variety of gels or polymers, for example,(poly)tetrafluoroethylene, (poly)vinylidenedifluoride, polystyrene,polycarbonate, or combinations thereof.

Suitable materials for forming the present reactors include, but are notlimited to, polymeric materials, ceramics (including aluminum oxide andthe like), glass, quartz, metals, composites, and laminates thereof.

In one embodiment, the substrate is glass. In other embodiments, thesubstrate is a polymeric material.

Polymeric materials will typically be organic polymers that arehomopolymers or copolymers, naturally occurring or synthetic,crosslinked or uncrosslinked. Specific polymers of interest include, butare not limited to, polyolefins such as polypropylene, polyimides,polycarbonates, polyesters, polyamides, polyethers, polyurethanes,polyfluorocarbons, polystyrenes, poly(acrylonitrile-butadiene-styrene)(ABS), acrylate and acrylic acid polymers such as polymethylmethacrylate, and other substituted and unsubstituted polyolefins, andcopolymers thereof.

The substrate and the cover plate may also be fabricated from a“composite,” i.e., a composition comprised of unlike materials. Thecomposite may be a block composite, e.g., an A-B-A block composite, anA-B—C block composite, or the like. Alternatively, the composite may bea heterogeneous combination of materials, i.e., in which the materialsare distinct from separate phases, or a homogeneous combination ofunlike materials. As used herein, the term “composite” is used toinclude a “laminate” composite. As used herein, the term “laminate”refers to a composite material formed from several different bondedlayers of identical or different materials. Other composite substratesinclude polymer laminates, polymer-metal laminates, e.g., polymer coatedwith copper, a ceramic-in-metal, or a polymer-in-metal composite.

The surfaces of the substrates and cover plates may be chemicallymodified to provide desirable chemical or physical properties, e.g., toreduce adsorption of molecular moieties to the interior walls of areaction chamber, and to reduce electro osmotic flow. For example, thesurface of a glass, a polymeric, or a ceramic substrate and/or coverplate may be coated with or functionalized to contain electricallyneutral molecular species, zwiterrionic groups, hydrophilic orhydrophobic oligomers or polymers, etc. With polyimides, polyamides, andpolyolefins having reactive sites or functional groups such as carboxyl,hydroxyl, amino and haloalkyl groups (e.g., polyvinyl alcohol,polyhydroxystyrene, polyacrylic acid, polyacrylonitrile, etc.), or withpolymers that can be modified so as to contain such reactive sites orfunctional groups, it is possible to chemically bond groups to thesurface that can provide a variety of desirable surface properties. Amodified substrate is polyimide functionalized so as to containsurface-bound water-soluble polymers such as polyethylene oxide (PEO),which tends to reduce unwanted adsorption and minimize nonspecificbinding in DNA amplification and other methodologies involvinghybridization techniques. The substrate surface may also beadvantageously modified using surfactants (e.g., polyethylene oxidetriblock copolymers such as those available under the tradename“Pluronic,” polyoxyethylene sorbitan, or “TWEEN”), natural polymers(e.g., bovine serum albumin or “BSA”), or other moieties that providethe desired surface characteristics, particularly in reducing thesorption of biomolecules such as nucleic acids or proteins.

It should also be emphasized that different regions of a singlesubstrate may have chemically different surfaces. For example, thereaction chamber may have one interior surface that is coated orfunctionalized, e.g., with polyethylene oxide or the like, while anotherinterior surface of the reaction chamber may not be coated orfunctionalized. In this way, different components and features presentin the same substrate may be used to conduct different chemical orbiochemical processes, or different steps within a single chemical orbiochemical process.

The substrate may be a thermally conductive material with a thermalconductivity greater than about 0.1 W/mK, or greater than about 0.5W/mK, or greater than about 1 W/mK. This allows for fast heat transferduring the rapid heating and cooling cycles.

In one embodiment, the substrate is a thermally conductive polypropylenewith a thermal conductivity greater than about 1 W/mK. Thermallyconductive polypropylenes typically include materials that act asheating elements. Suitable heat conducting materials may include, forexample, iron, nickel, cobalt, chromium; carbon steel fibers, magneticstainless steel fibers, nickel fibers, ferromagnetic coated electricallyconductive fibers, ferromagnetic coated electrically nonconductivefibers, and alloys thereof.

In one embodiment, the substrate is heated to raise the temperature ofthe reactor. In another embodiment, the substrate is cooled to lower thetemperature of the reactor. In yet another embodiment, the substrate isboth heated and then cooled to regulate the temperature of the reactor.

In one embodiment, the cover plate is glass.

The Buffer Layer

In the reactor, a buffer layer is used between the substrate and thecover plate. The buffer layer should have good adhesion to the substrateand the cover plate. The buffer layer should also be impenetrable by theliquid used in the sample. The buffer layer should be able to withstandrepeated cycling between 4° C. through 95° C. for extended periods oftime (e.g., 1-2 hours). The buffer layer should also not interfere withthe PCR process and the detection system.

A variety of buffer layers may be used, although any buffer layerselected should be capable of withstanding the forces generated duringprocessing of any sample materials located in the reaction chamber, forexample, forces developed during distribution of the sample materials,forces developed during thermal processing of the sample materials, etc.Those forces may be large where, for example, the processing involvesthermal cycling. In one embodiment, the buffer layer used in connectionwith the sample processing devices should exhibit low fluorescence andbe compatible with the processes and materials to be used in connectionwith PCR.

In one embodiment, the buffer layer may exhibit sealant and/or adhesiveproperties. Such buffer layers may be more amenable to high volumeproduction of sample processing devices since they typically do notinvolve the high temperature bonding processes used in melt bonding, nordo they present the handling problems inherent in use of liquidadhesives, solvent bonding, ultrasonic bonding, and the like.

In one embodiment, the buffer layer may include materials which ensurethat the properties of the buffer layer are not adversely affected bywater. For example, the buffer layer should not lose adhesion, losecohesive strength, soften, swell, or opacify in response to exposure towater during sample loading and processing. Also, the buffer layershould not contain any components which may be extracted into waterduring sample processing, thus possibly compromising the deviceperformance.

Furthermore, the buffer layer can be a single material or a combinationor blend of two or more materials. The buffer layer may result from, forexample, solvent coating, screen printing, roller printing, meltextrusion coating, melt spraying, stripe coating, or laminatingprocesses. A buffer layer can have a wide variety of thicknesses as longas it meets exhibits the above characteristics and properties. In orderto achieve maximum bond fidelity and, if desired, to serve as apassivating layer, the buffer layer should be continuous and free frompinholes or porosity.

Any adhesive composition known in the art can be applied as the bufferlayer. Suitable adhesive compositions are described in, for example,“Adhesion and Bonding,” Encyclopedia of Polymer Science and Engineering,Vol. 1, pp. 476-546, Interscience Publishers, Second Ed., 1985. In oneembodiment, the adhesive compositions are water-impermeable. Suitablewater impermeable adhesives include, for example, natural rubber latexbased adhesives, synthetic rubber based adhesives, silicon basedadhesives, and hot-melt adhesives. Many other adhesives can also be usedfor purposes of the present invention the particular choice beingdependent on the character of the two surfaces to be bound to eachother, the circumstances under which the bonding is to be accomplishedand the intended use of the resulting products. A thorough discussion ofadhesives can be found in Ullmann's Encyclopedia of IndustrialChemistry, VCH Verlagsgesellschaft GmbH, Germany, 1985, Vol. A1, atpages 221-267 and Encyclopedia of Chemical Technology, Fourth Ed., JohnWiley & Sons, NYC, 1991, Vol. 1, at pages 445-466. Curable adhesives arealso be used. However, contact, pressure sensitive, rubber based,emulsion, hot melt, natural product, polyurethane, acrylic, epoxy,phenolic, and polyimide adhesives may also be used.

Suitable classes of sealant compositions may also include, for example,polyurethanes, polyisobutylenes, butyl rubbers, elastomers, epoxys,natural and synthetic rubber, silicones, polysulfides, acrylates, andcombinations thereof. Sealant compositions may include polar and/orreactive groups (e.g., silane, urethane, ester, mercapto, andcombinations thereof) to provide sufficient covalent, and/or polar(e.g., hydrogen) bonding with the target substrates (e.g., glass andplastic).

In one embodiment, the buffer layer may be composed of hydrophobicmaterials. In one embodiment, the buffer layer may be composed ofsilicone materials.

In one embodiment, a silicon sealant is used. Silicone sealantstypically include a mixture of a silicone polymer, one or more fillers,a crosslinking component such as a reactive silane, and a catalyst. Thesilicone polymer has a siloxane backbone and includes pendant alkyl,alkoxy, or acetoxy groups. Such groups are hydrolyzed to silanol groupswhich form larger chains by condensation. The silicone sealants may beapplied by means of a caulking gun, a spatula, or other suitable methodand are cured by exposure in moist air. The silicone sealants have lowshrinkage characteristics and may be applied and used over a widetemperature range. Room Temperature Vulcanizing (RTV) silicone rubbersealants are particularly useful due to their mild curing conditions.Suitable Room Temperature Vulcanizing (RTV) silicone rubber sealantsinclude, for example, a one component RTV rubber (KE3475, Shin-EtsuChemical Co., Ltd., Japan) and the one-part moisture cure RTV (SE 9120,Dow Corning Corporation, Midland, Mich., USA).

In addition to moisture curing silicon sealant materials,radiation-curable silicon sealants may also be used. A suitableultraviolet radiation-curable silicone sealant composition typicallycomprises (i) an organopolysiloxane containing radiation-sensitivefunctional groups and (ii) a photoinitiator. Examples ofradiation-sensitive functional groups include acryloyl, methacryloyl,mercapto, epoxy, and alkenyl ether groups. The type of photoinitiatordepends on the nature of the radiation-sensitive groups in theorganopolysiloxane. Examples of photoinitiators may includediaryliodonium salts, sulfonium salts, acetophenone, benzophenone, andbenzoin and its derivatives. A particularly useful type of unsaturatedorganosilicon compound has at least one aliphatically unsaturatedorganic radical attached to silicon per molecule. The aliphaticallyunsaturated organosilicon compounds include silanes, polysilanes,siloxanes, silazanes, as well as monomeric or polymeric materialscontaining silicon atoms joined together by methylene or polymethylenegroups or by phenylene groups.

The buffer layer may also be selected from the class of siliconematerials, based on the combination of silicone polymers and tackifyingresins, as described in, for example, “Silicone Pressure SensitiveAdhesives,” Handbook of Pressure Sensitive Adhesive Technology, 3rdEdition, pp. 508-517. Silicone pressure sensitive adhesives are knownfor their hydrophobicity, their ability to withstand high temperatures,and their ability to bond to a variety of dissimilar surfaces.

Some suitable compositions may be described in PCT Patent ApplicationPublication No. WO 00/68336. Other suitable compositions may be based onthe family of silicone-polyurea based pressure sensitive adhesives. Suchcompositions are described in U.S. Pat. No. 5,461,134; U.S. Pat. No.6,007,914; PCT Patent Application Publication No. WO 96/35458; PCTPatent Application Publication No. WO 96/34028; and PCT PatentApplication Publication No. WO 96/34029. Such pressure sensitiveadhesives are based on the combination of silicone-polyurea polymers andtackifying agents. Tackifying agents can be chosen from within thecategories of functional (reactive) and nonfunctional tackifiers asdesired. The level of tackifying agent or agents can be varied asdesired so as to impart the desired tackiness to the adhesivecomposition. For example, in one embodiment, the pressure sensitiveadhesive composition may be a tackified polydiorganosiloxane oligureasegmented copolymer including (a) soft polydiorganosiloxane units, hardpolyisocyanate residue units, wherein the polyisocyanate residue is thepolyisocyanate minus the —NCO groups, optionally, soft and/or hardorganic polyamine units, wherein the residues of isocyanate units andamine units are connected by urea linkages; and (b) one or moretackifying agents (e.g., silicate resins, etc.).

In some embodiments, the barrier layer may be, for example, a single ordouble-sided water-impermeable adhesive tape. In other embodiments, thebarrier layer may be, for example, a gasket coated on one or both sideswith water-impermeable adhesive. In other embodiments, the barrier layermay be, for example, a water-impermeable laminate material.

Fabrication

The substrate can be fabricated using any convenient method, including,but not limited to, micromolding and casting techniques, embossingmethods, surface micro-machining and bulk-micromachining. The lattertechnique involves formation of microstructures by etching directly intoa bulk material, typically using wet chemical etching or reactive ionetching. Surface micro-machining involves fabrication from filmsdeposited on the surface of a substrate.

Although the foregoing discussion has used DNA as a nucleic acid, itwould be obvious to a person of reasonable skill in the art to apply themethods disclosed herein to other nucleic acids, including RNA sequencesor combinations of RNA and DNA sequences.

It is to be understood that certain descriptions of the presentinvention have been simplified to illustrate only those elements andlimitations that are relevant to a clear understanding of the presentinvention, while eliminating, for purposes of clarity, other elements.Those of ordinary skills in the art, upon considering the presentdescription of the invention, will recognize that other elements and/orlimitations may be desirable in order to implement the presentinvention. However, because such other elements and/or limitations maybe readily ascertained by one of ordinary skill upon considering thepresent description of the invention, and are not necessary for acomplete understanding of the present invention, a discussion of suchelements and limitations is not provided herein.

EXAMPLES

The following Example is illustrative of the above invention. Oneskilled in the art will readily recognize that the techniques andreagents described in the Example suggest many other ways in which thepresent invention could be practiced.

Example 1

This example illustrates the fabrication of a microarray reactor for thequantitative analysis of nucleic acids using a polymerase chain reaction(PCR) process and an evanescent wave detection technique.

The reaction chamber is made of a glass cover plate and a thermallyconductive polypropylene substrate. The interior surface of the glasscover plate is chemically modified to reduce the adsorption offluorescent substances and other contaminants. The target nucleic acidprobes are tethered to the interior surface of the glass cover plate ina known, two-dimensional pattern. The glass cover plate is alsotransparent and suitable for an evanescent wave detection technique.

The thermally conductive polypropylene substrate with an interior cavityis fabricated using a molding method. An inlet and an outlet areincorporated into the substrate. The glass cover plate and thepolypropylene substrate are assembled and sealed together by a bufferlayer to form a reactor. After the sample is loaded, both the inlet andthe outlet are sealed with a rubber plug.

To prevent liquid leakage of the reactor with thermal cycling, a bufferlayer is used between the substrate and the cover plate. A curablesilicone rubber (KE3475 from Shin-Etsu Chemical Co., Ltd., Japan) isused as a buffer layer. This silicone rubber is water-impenetrable andable to withstand temperatures between 4° C. through 95° C. Thissilicone rubber will also not interfere with the PCR process or exhibitlow fluorescence after curing. To prevent damage to the glass coverplate, the polypropylene substrate, and the immobilized target probe thesilicon rubber is a room temperature vulcanizing (RTV) material.

The sample and various analytical reagents and reactants are introducedinto the reaction chamber from an external source through inlet port.The outlet port acts as a blowhole when fluid is introduced in throughinlet port. After the sample and various analytical reagents andreactants is added, a set of rubber plugs with the proper size,hardness, and chemical resistance are used to seal the inlet port andoutlet port of the reaction chamber.

When using the reactor for nucleic acid detection, the reactor and thereagent inside is heated and cooled down by a PCR temperature cyclingprogram. For example, a semi-conductor cooler is used forheating/cooling the substrate made of a thermally conductivepolypropylene material. During the PCR process, the target DNA in thechamber is exponential amplified and the amplified DNA products arehybridized to the target probe tethered on the interior surface of theglass cover plate at annealing/extending step in every amplificationcycle. The glass cover plate is suitable for fluorescent detection byevanescent wave. The glass cover plate may be made, for example, of K9optical glass with refractive index larger than the refractive index ofthe PCR/hybridization buffer inside the reactor. A fluorescent molecule,for example, CY5, may be used for PCR primer labeling. CY5 is excitedmaximally at 649 nm and emits maximally at 670 nm.

The invention has been described with reference to various specificembodiments and techniques. However, it should be understood that manyvariations and modifications may be made while remaining within thespirit and scope of the invention.

1. A reactor for the quantitative analysis of target nucleic acids,comprising: a substrate having a first planar opposing surface and asecond planar opposing surface, the substrate having a cavity; a bufferlayer arranged over the first planar surface of the substrate; a coverplate arranged over the buffer layer, the cover plate in combinationwith the cavity and buffer layer defining a reaction chamber; and atleast one inlet port and at least one outlet port communicating with thereaction chamber through the substrate enabling the passage of fluidfrom an external source into and through the reaction chamber, andthereby defining a fluid flow path; wherein the quantitative analysis oftarget nucleic acids uses a polymerase chain reaction (PCR) process andan evanescent wave detection technique.
 2. The reactor of claim 1,wherein the substrate, the buffer layer, and the cover plate are eachindependently comprised of a chemically inert material that is thermallystable and resistant to contamination.
 3. The reactor of claim 1,wherein the substrate and the cover plate are each independently aglass, a metal, a ceramic, a composite, a polymeric material, or acombination or laminate thereof.
 4. The reactor of claim 3, wherein thepolymeric material is a polyimide, a polycarbonate, a polyester, apolyamide, a polyether, a polyurethane, a polyfluorocarbon, apolystyrene, a poly(acrylonitrile-butadiene-styrene), a polymethylmethacrylate, a polyolefin, or a copolymer thereof.
 5. The reactor ofclaim 3, wherein substrate is a thermally conductive polypropylene. 6.The reactor of claim 1, wherein substrate has a thermal conductivitygreater than about 1 W/mK.
 7. The reactor of claim 1, wherein the bufferlayer is a water-impermeable sealant.
 8. The reactor of claim 7, whereinthe water-impermeable sealant is a room temperature vulcanizing siliconerubber.
 9. The reactor of claim 3, wherein the cover plate is a glassplate.
 10. A method for the quantitative analysis of target nucleicacids, comprising: (a) introducing into a reactor up to a sample fluidcontaining one or more target nucleic acids, one or more fluorescentlytagged primers, one or more optionally fluorescently tagged dNTPs, athermostable nucleic acid polymerase, and a buffer, the reactorcomprising: a substrate having a first planar opposing surface and asecond planar opposing surface, the substrate having a cavity; a bufferlayer arranged over the first planar surface of the substrate; a coverplate arranged over the buffer layer, the cover plate in combinationwith the cavity and buffer layer defining a reaction chamber; and atleast one inlet port and at least one outlet port communicating with thereaction chamber through the substrate enabling the passage of fluidfrom an external source into and through the reaction chamber, andthereby defining a fluid flow path; (b) applying a motive force to movethe sample fluid along the flow path into the reaction chamber; (c)sealing the at least one inlet port and the at least one outlet port;(d) heating the sample fluid in the reaction chamber to separate the oneor more double-stranded target nucleic acids into single-stranded targetnucleic acids; (e) cooling the sample to allow hybridization of the oneor more fluorescently tagged primers to the single-stranded targetnucleic acids and replication of the single-stranded target nucleicacids by the thermostable nucleic acid polymerase; and (f) repeatingsteps (d) and (e) to achieve the desired degree of amplification; (g)activating one or more fluorescence responses from one or morefluorescently tagged amplicons hybridized to the one or more targetnucleic acids; and (h) detecting the one or more fluorescence responsesfor a quantitative analysis of the one or more target nucleic acids,wherein the activating of the one or more fluorescence responses is byusing an evanescent wave of a predetermined wavelength.
 11. The methodof claim 10, wherein the quantitative analysis of nucleic acids uses apolymerase chain reaction (PCR) process.
 12. The method of claim 10,wherein the motive force comprises a means for applying a voltagedifferential or a pressure differential.
 13. The method of claim 10,wherein the substrate and the cover plate are each independently aglass, a metal, a ceramic, a composite, a polymeric material, or acombination or laminate thereof.
 14. The method of claim 13, wherein thepolymeric material is a polyimide, a polycarbonate, a polyester, apolyamide, a polyether, a polyurethane, a polyfluorocarbon, apolystyrene, a poly(acrylonitrile-butadiene-styrene), a polymethylmethacrylate, a polyolefin, or a copolymer thereof.
 15. The method ofclaim 13, wherein substrate is a thermally conductive polypropylene. 16.The method of claim 10, wherein the substrate has a thermal conductivitygreater than about 1 W/mK.
 17. The method of claim 10, wherein thebuffer layer is a water-impermeable sealant.
 18. The method of claim 17,wherein the water-impermeable sealant is a room temperature vulcanizingsilicone rubber.
 19. The method of claim 13, wherein the cover plate isa glass plate.
 20. A reactor for the quantitative analysis of targetnucleic acids, comprising: a substrate having a first planar opposingsurface and a second planar opposing surface, the substrate having acavity; a buffer layer arranged over the first planar surface of thesubstrate; a cover plate arranged over the buffer layer, the cover platein combination with the cavity and buffer layer defining a reactionchamber; and at least one inlet port and at least one outlet portcommunicating with the reaction chamber through the substrate enablingthe passage of fluid from an external source into and through thereaction chamber, and thereby defining a fluid flow path; wherein thequantitative analysis of target nucleic acids uses a polymerase chainreaction (PCR) process and an evanescent wave detection technique,wherein the substrate is a polymeric material, the buffer layer is awater-impermeable sealant, and the cover plate is a glass plate, andwherein the substrate has with a thermal conductivity greater than about1 W/mK.